Cloning, expression and characterization of Rigidoporus vinctus alcohol dehydrogenase
| Autor: | Tzeng, Shiun-Jye, 曾薰頡 |
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| Rok vydání: | 2014 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 102 Alcohol dehydrogenases are widely distributed enzymes and have been found in many microorganisms and in fungal, plant, and animal cells. ADHs catalyze the reversible oxidation of alcohols to the corresponding aldehydes or ketones. In this study, a full length cDNA (1035 bp) encoding a putative ADH from Rigidoporus vinctus was cloned by polymerase chain reaction (PCR). The coding region of RvADH encodes 344 amino acids. To further characterize the RvADH, the coding region was subcloned into an expression vector pET-20b(+), which can be induced by IPTG, and transformed into E. coli Rosetta (DE3) pLysS. The RvADH is 38 kDa, and has 6 his tag. The expression of the RvADH was purified by Ni2+-nitrilotriacetic acid Sepharose superflow column. Subsequently, the purified enzyme was analyzed by 12% SDS-PAGE. Furthermore, the enzyme activity and kinetics were determined by NAD+ reduction assay while using ethanol as substrate. The KM value is 0.79 mM; Vmax is 0.005 mM per min and kcat is 20.6 per min. The enzyme is more stable in pH 9. The enzyme has a half-life of 3.4 min at 50 °C and kd value is 9.62 #westeur024# 10-2 min-1. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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